1. Field of the Invention
The present invention relates generally to neurology and, more particularly, to assays, such as immunoassays, for screening for compounds that specifically alter the production of various isoforms of Aβ.
Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death. AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States. AD has been observed in all races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms or course is currently known.
The brains of individuals with AD exhibit characteristic lesions termed senile plaques and neurofibrillary tangles. Large numbers of these lesions are generally found in patients with AD in several areas of the human brain important for memory and cognitive function. Smaller numbers of these lesions in a more restricted anatomical distribution are sometimes found in the brains of aged humans who do not have clinical AD. Senile plaques and vascular amyloid deposits (amyloid angiopathy) also characterize the brains of individuals beyond a certain age with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). The principal chemical constituent of the senile plaques and vascular amyloid deposits characteristic of AD and the other disorders mentioned above is a protein designated the amyloid-β peptide (Aβ) or sometimes βAP, AβP or β/A4. Aβ was first purified and a partial amino acid sequence reported in Glenner and Wong (1984) Biochem. Biophys. Res. Commun.120:885-890. The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Pat. No. 4,666,829. Forms of Aβ having amino acids beyond number 40 were first reported by Kang et al. (1987) Nature 325:733-736.
Roher et al. (1993) Proc. Natl. Acad. Sci. USA 90:10836-840 showed that Aβ(1-42) is the major constituent in neuritic plaques, including significant amounts of isomerized and racemized aspartyl residues as their NH2-termini. The authors also reported that Aβ(17-42) (p3(42)) predominates in diffuse (early) plaques, whereas Aβ(1-40) is the major constituent in the meningeal vessel deposits, comprising 60% of the total Aβ in those vessels. Iwatsubo et al. (1994) Neuron 13:45-53 showed that Aβ42(43)-containing senile plaques are the major species of senile plaques in sporadic AD brains. Iwatsubo et al. (1995) Annals of Neurology 37:294-299 and Lemere et al. (1996) Neurobiology of Disease 3:16-32 reported that Aβ42(43) is the major constituent of senile plaques in Down's syndrome brains and is the initially deposited Aβ species in the development of AD-type neuropathological legions in these patients. In addition, Gravina et al., (1995) J. Biol. Chem. 270:7013-7016 reported both biochemical and immunocytochemical evidence that Aβ42(43) peptides were the most abundant constituents of senile plaques in AD brains and exceeded the amounts of Aβ40 peptides in such plaques.
Molecular biological and protein chemical analyses conducted during the last several years have shown that Aβ is a small fragment of a much larger precursor protein, referred to as the β-amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans. Knowledge of the structure of the gene encoding APP has demonstrated that Aβ arises as a peptide fragment that is cleaved from the carboxy-terminal end of APP by as-yet-unknown enzymes (proteases). The precise biochemical mechanism by which the Aβ fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls of cerebral and meningeal blood vessels is currently unknown. Importantly, Haass et al. (Nature 359:322-325) and Seubert et al. ((1992) Nature 359:325-327) discovered that essentially all cells expressing the APP gene normally secrete an array of Aβ peptides, and these peptides can readily be detected and assayed in cell culture fluid (conditioned media) and human biological fluids such as plasma and cerebrospinal fluid. It has subsequently been shown that these fluids contain both the more abundant Aβ40-ending peptides and the less abundant Aβ42(43)-ending peptides (Dovey et al. (1993) Neuroreport 4:1039-1042 and Vigo-Pelfrey et al. (1993) J. Neurochem. 61:1965-68)
Several lines of evidence indicate that progressive cerebral deposition of Aβ plays a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades (for review, see Schenk (1995) J. Med. Chem. 38:4141-4154, Selkoe (1994) J. Neuropath. and Exp. Neurol. 53:438-447 and Selkoe (1991) Neuron 6:487). One of the most important lines of evidence is the discovery in 1991 that missense DNA mutations in the APP gene at amino acid 717 of the 770-amino acid isoform of APP can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD (Goate et al. (1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature 353:844-846; and Murrell et al. (1991) Science 254:97-99). Suzuki et al. (1994) “An increased percentage of long amyloid β-protein secreted by familial amyloid β-protein precursor (βAPP717) mutants,” Science 264:1336-1340 subsequently showed that, compared to normal individuals, the 717 mutation causes a higher relative production of the Aβ(1-42) peptide. In addition, a double mutation changing lysine670-methionine671 to asparagine670-leucine671 (with reference to the 770 isoform of APP) was reported in a Swedish family with familial AD in 1992 (Mullan et al. (1992) Nature Genet 1:345-347) and is referred to as the Swedish APP variant.
Genetic linkage analyses have demonstrated that the aforementioned mutations are the specific molecular cause of AD in the members of such families that carry these mutant APP genes. In addition, a mutation at amino acid 693 of the 770-amino acid isoform of APP has been identified as the cause of the Aβ deposition disease, Hereditary Cerebral Hemorrhage With Amyloidosis Dutch type (HCHWA-D), and a mutation from alanine to glycine at amino acid 692 appears to cause the phenotype of AD in some family members and the phenotype of HCHWA-D in others. The discovery of these APP mutations in genetically based cases of AD argues that genetic alteration of APP and subsequent deposition of its Aβ fragment can cause AD.
Recently, evidence has accumulated suggesting that Aβ(42) plays the key role in the process of senile plaque formation in AD. First, in vitro data demonstrate that Aβ(42) accelerates the formation of Aβ fibrils (and thus senile plaques) by a nucleation dependent mechanism (Jarrett et al. (1993) Biochemistry 32:4693-4697). Second, while accounting for ≦10% of total Aβ secreted from cells (roughly 90% is Aβ(40) (Dovey et al. (1993) Neuroreport 4:1039-1042; Asami-Odaka et al. (1995) “Long amyloid β-protein secreted from wild-type human neuroblastoma IMR-32 cells.” Biochemistry 34:10272-10278), Aβ(42) is the major plaque component (Kang et al. (1987) Nature 325:733-736; Iwatsubo et al. (1994) Neuron 13:45-53; Iwatsubo et al. (1995) Ann. Neurol. 37:294-299; Gravina et al. (1995) J. Biol. Chem. 270:7013-7016; Lemere et al. (1996) Neurobiology of Disease 3:16-32). Furthermore, all 3 early onset familial AD genes identified to date have been shown to lead to an increase in cellular secretion of Aβ(42). Only the Swedish APP missense mutation increases the secretion of both Aβ(40) and Aβ(42) peptides (Dovey et al. (1993) Neuroreport 4:1039-1042, whereas the APP717 mutations and the presenilin mutations appear not to increase Aβ(40) peptides (Suzuki et al., (1994) Science 264:1336-1340; Scheuner et al. (1995) Neurosci. Abstracts in press). Thus, the longer Aβ(42) peptide appears to be a prime target for therapeutic intervention. However, none of the proteases involved in the major steps of APP processing have been definitively identified, including γ-secretase, the protease which generates the C-terminus of Aβ. It has generally been assumed that the same protease(s) generate both Aβ(40) and A(42) and it has been shown that both forms share a common secretory mechanism which involves acidic intracellular compartments such as the late Golgi or early endosomes (Koo and Squazzo (1994) J. Biol. Chem. 269:17386-17389; Asami-Odaka et al. (1995) Biochemistry 34:10272-10278). Recently, Higaki et al. ((1995) Neuron, 14:651-659) have shown that the Calpain inhibitor, MDL 28170, inhibits the production of both total Aβ and total p3 and leads to an accumulation of their respective 12 kDa and 10 kDa APP precursor fragments in treated cells. These data suggest that the compound directly inhibits at least some form of γ-secretase although no data are provided as to what specific form of Aβ and p3 are affected.
Despite the progress which has been made in understanding the underlying mechanisms of AD, there remains a need for assays to identify candidate compounds for preventing or treating the disease.